Therapeutic bacteriophage compositions

ABSTRACT

The present invention provides methods of designing panels of bacteriophages as therapeutic compositions against bacterial infections. The present invention also provides panels of bacteriophages for use in the prevention or treatment of bacterial infections.

This patent application claims priority to GB 1207910.9 filed on 4 May2012, and to GB 1218083.2 filed on 9 Oct. 2012, which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods for preparing panels ofbacteriophages (whether as a premixed cocktail or for mixing prior touse).

BACKGROUND TO THE INVENTION

Antibiotic resistance is now seen as one of the major challenges facingmodern medicine. Given the shortage of novel antibiotics, a number ofalternative approaches are being investigated, including the use ofbacteriophages as therapeutic agents (Harper, Anderson & Enright,Therapeutic Delivery (2011), 2, 935-947; Hausler T, Viruses vs.Superbugs: A Solution to the Antibiotics Crisis? (2006) MacMillan, N.Y.

Bacteriophages (often known simply as “phages”) are viruses that growwithin bacteria. The name translates as “eaters of bacteria” andreflects the fact that as they grow most bacteriophages kill thebacterial host as the next generation of bacteriophages is released.Early work with bacteriophages was hindered by many factors, one ofwhich was the widespread belief that there was only one type ofbacteriophage, a non-specific virus that killed all bacteria. Incontrast, it is now understood that the host range of bacteriophages(the spectrum of bacteria they are capable of infecting) is often veryspecific. This specificity, however, has the disadvantage that it isdifficult to achieve breadth of adequate bacteriophage efficacy acrossbacterial target species/strains. There is therefore a need in the artfor methods of identifying improved combinations of bacteriophageshaving effective targeting capability in relation to bacterialspecies/strains—see, for example, Pirsi, The Lancet (2000) 355, 1418.For these reasons, examples of phage compositions demonstrating soundclinical efficacy are very limited. By way of example, reference is madeto Applicant's successful clinical trials (veterinary and human)conducted with a panel of bacteriophages that target Pseudomonasaeruginosa—see Wright et al, Clinical Otolaryngology (2009) 34, 349-357.There is therefore a need in the art to develop further panels ofbacteriophages that have optimal clinical applicability.

In particular, there is a need in the art to design panels of two ormore bacteriophages that target the same bacterial host species/strain,wherein said panel of bacteriophage provide adequate efficacy against abacterial target species/strain when compared to the individual efficacyof said bacteriophage against said bacterial target species/strain. Inthis regard, it is necessary that the bacteriophage members of the panelwork well together in a combination (e.g. the panel demonstratesequivalent or improved efficacy vis-à-vis the individual membersthereof).

The present invention addresses one or more of the above problems.

SUMMARY OF THE INVENTION

The present invention solves the above described problems by providingmethods for designing panels of bacteriophages, as specified in theclaims. The present invention also provides panels of bacteriophages anduses thereof, as specified in the claims.

In one aspect, the present invention provides a method for designing anoptimal therapeutic panel of bacteriophages (comprising two or morebacteriophages). Said method includes assaying the activity ofindividual bacteriophages in liquid cultures of a target bacterialspecies/strain to determine the kinetics of bacterial growth, togetherwith the development and specificity of resistance developed by thebacterial target in said culture. The method further includesdetermining the efficacy of bacteriophage panels in said culture, andthus identifying an advantageous bacteriophage panel for use against thetarget bacterial species/strain.

Bacteriophages that infect the same bacterial species/strain may employsimilar mechanisms of infection, meaning that resistance of thebacterial species/strain to one bacteriophage confers cross-resistanceto other bacteriophages—see Gill & Hyman, Curr. Pharm. Biotech. (2010)11, 2-14; and Guidolin & Manning, Eur. J. Biochem (1985) 153, 89-94.Clearly this is undesirable. Additionally, the present inventors haveunexpectedly identified that bacteriophages can be antagonistic towardsone another when targeting a given bacterial species/strain, therebylimiting the effect of co-infecting bacteriophages.

In one aspect, the present invention therefore provides a method fordesigning a panel of bacteriophages (comprising two or morebacteriophages), which minimises target bacterial species/strainresistance to each of said individual bacteriophages (i.e.cross-resistance) in the panel, and/or antagonism between saidbacteriophages when targeting the bacterial species/strain. Said methodemploys a process of measuring bacterial target growth characteristicsand/or bacteriophage growth characteristics when present in liquidcultures of their host (target) bacteria, following by selection of atherapeutic panel of bacteriophages.

Individual lytic bacteriophages may be tested in plaque assay and/or inliquid (broth) culture with their bacterial host—both tests arepreferably employed (e.g. one test may be performed sequentially orprior to the next, or both may be performed substantiallysimultaneously). Those that show efficient killing of the bacterial hostin these two systems are not necessarily identical. By way of example,plaque assay is a complex dynamic process (Abedon & Yin, Methods Mol.Biol. (2009) 501, 161-174), whereas broth culture provides a lessstructured environment in which to monitor lysis (killing) of thebacterial host.

Bacterial numbers in such liquid cultures may be monitored directly byviable count of an aliquot of the culture medium. Alternatively,bacterial numbers may be measured by assaying the optical density of theculture. By way of example, plate reader systems allow such cultures tobe monitored directly in high throughput systems, typically with opticaldensity measured at 600 nm.

In liquid cultures not treated with bacteriophage, bacterial numbersincrease over several hours, eventually slowing as nutrients areexhausted and bacterial numbers reach a maximum level. When treated withbacteriophage, bacterial numbers typically increase for a short timethen decline rapidly. However, when treated with a single (e.g. a first)bacteriophage (or a mixture of bacteriophages where cross-resistanceoccurs) after several hours resistant bacteria start to appear andbacterial numbers again increase.

By sampling these resistant bacteria and assaying the effect ofdifferent bacteriophages (e.g. second and/or third bacteriophages, etc.)on them, bacteriophages (e.g. second and/or third differentbacteriophages, etc.) are identified where bacterial resistance to onephage (e.g. the first phage) does not confer resistance to others phages(e.g. second and/or third different bacteriophages, etc.)—referred toherein as a lack of cross-resistance to phage. The selection and use ofbacteriophage panels comprising bacteriophages that demonstrate a lackof cross-resistance to a target bacterial species/strain is highlydesirable in bacteriophage panels designed for use as an anti-microbialtherapeutic.

Once a panel of bacteriophages (having desired characteristics ashereinbefore identified), the panel may then be tested in liquidculture. Surprisingly, some mixtures of individual bacteriophages do notnecessarily produce additive effects. In particular, antagonism occurswhere the effects of combined phages are less effective at reducingbacterial numbers than are achieved with the corresponding individualbacteriophages in isolation. Monitoring the efficacy of such mixtures inreducing bacterial numbers in liquid culture provides a means ofidentifying such antagonistic combinations, which are considerednon-optimal for further development as candidate therapeutics.

Methods for determining growth of bacteria (such as a target bacterialspecies or strain) are known in the art. By way of example, growth canbe determined of a target bacterial species or strain growing in aculture, such as a liquid culture. In this regard, as the bacteriamultiply and increase in number, the optical density of the liquidculture increases (due to the presence of an increasing number ofbacterial cells). Thus, an increase in optical density indicatesbacterial growth. Optical density may be measured at 600 nm (OD₆₀₀). Forexample, optical density at 600 nm can be determined within the wells ofa multi-well plate (e.g. a 96-well plate) using an automated platereader (for example a BMG Labtech FLUOstar Omega plate reader).

Growth of a target bacterial species or strain can be determined and/ormonitored over a defined time period (for example, at least 2, 4, 8, 12,16, 20, 24, 36 or 48 hours).

In some embodiments, a time period may be defined as starting from theaddition of one or more different bacteriophages to a target bacterialspecies or strain. Alternatively, a time period may be defined asstarting at a predetermined point after the addition of one or moredifferent bacteriophages to a target bacterial species or zo strain (forexample, starting at least 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 or 12hours after).

Methods of determining if a bacteriophage or combination ofbacteriophages retards growth (i.e. effects growth retardation) of agiven population of bacteria (for example, a target bacterial species orstrain, as specified in the claims; or a resistant culture, as specifiedin the claims) are known in the art.

As a bacteriophage (or combination of bacteriophages) multiplies in hostbacteria, bacterial lysis occurs, killing bacteria and leading to adecrease in bacterial growth. A decrease in bacterial growth can includea decrease in the rate of growth (e.g. the rate at which the bacterialcell number increases), a cessation of growth (such that the bacterialcell number remains constant), or a decrease in the total bacterial cellnumber.

In one embodiment, growth retardation (i.e. when a bacteriophage orcombination of bacteriophages retards growth) means that bacterialgrowth in the presence of a given bacteriophage or combination ofbacteriophages is decreased as compared to bacterial growth of anequivalent population of bacteria (under the same or equivalentconditions) in the absence of said bacteriophage or combination ofbacteriophages.

Methods for determining bacterial growth are known in the art, asdescribed above. Thus, methods used to determine bacterial growth (e.g.through measurement of bacterial numbers) may also be used to determinegrowth retardation. Thus, by way of example, growth retardation may bedetermined at a specified time point or over a specified period of timefollowing addition of a bacteriophage or combination of bacteriophagesto a bacterial population (for example, at least 2, 4, 8, 12, 16, 20,24, 36 or 48 hours). By way of example, the specified period of time mayembrace the logarithmic phase of bacterial growth.

In one embodiment, wherein the invention provides a method of designinga panel of bacteriophages as a therapeutic composition against abacterial infection, as specified in any of claims 1-4, if a combinationof bacteriophages retards growth of zo the target bacterial species orstrain at least equal to the greatest growth retardation achievedindependently by any one of said two or more different bacteriophages,the combination is accepted as a panel of bacteriophages, and thebacteriophages which make up said combination are deemed to lackantagonism.

Methods for determining the development of bacterial resistance againsta bacteriophage or combination of bacteriophages are known in the art.By way of example, the development of bacterial resistance may bedetermined by monitoring bacterial growth in the presence of abacteriophage or combination of bacteriophages. Bacterial growth may bemonitored as described above. Thus, in the absence of bacterialresistance against the bacteriophage or combination of bacteriophages,growth retardation (as described above) may be observed. As bacterialresistance develops, the effects of growth retardation are overcome andbacterial growth increases. The development of bacterial resistance maybe determined by monitoring bacterial growth for a specified period oftime, as described above (for example, at least 2, 4, 8, 12, 16, 20, 24,36 or 48 hours).

Determining the development of bacterial resistance can also allow theidentification of combinations of bacteriophages wherein bacterialresistance to one bacteriophage does not confer resistance to anotherbacteriophage in the combination (referred to as a lack ofcross-resistance, as described above).

Thus, in one embodiment, wherein the invention provides a method ofdesigning a panel of bacteriophages as a therapeutic composition againsta bacterial infection, as specified in claim 5, below, if said secondbacteriophage retards growth of the first resistant bacterial culture,the target bacterial species or strain is deemed to lackcross-resistance to the combination of said first and secondbacteriophages.

In another embodiment, wherein the invention provides a method ofdesigning a panel of bacteriophages as a therapeutic composition againsta bacterial infection, as specified in claim 7, below, if said thirdbacteriophage retards growth of the second resistant bacterial culture,the target bacterial species or strain is deemed to lackcross-resistance to the combination of at least said second and thirdbacteriophages; preferably, the target bacterial species or strain isdeemed to lack cross-resistance to the combination of said first, secondand third bacteriophages.

EXAMPLES

A mixture for in vivo use was developed against the PAK strain ofPseudomonas aeruginosa. The stages of this development exemplify thestages of the invention.

Initial Screening:

The Pseudomonas aeruginosa strain PAK is used in studies of mouse lunginfection, using an inserted luminescent reporter gene to identifynon-invasively the sites and levels of infection.

To identify bacteriophages for a therapeutic bacteriophage mix for useagainst the PAK strain, bacteriophages grown on permissive host strainswere then tested against the PAK strain by spot testing on bacteriallawns, enumerative plaque assay and broth culture using a plate readerassay system. The plate reader monitors intensively the optical densityof a broth culture containing bacteriophages with a suitable host in amulti-well plate format. This latter method allows detailed kinetics ofthe infection process to be evaluated.

Screening of individual bacteriophages by plaque assay and in liquidculture produced the results shown in Table 1. [MOI=multiplicity ofinfection (ratio of infecting bacteriophage to bacterial host cells)].

The marked discrepancy between the poor plaque formation bybacteriophage BCP37 and its efficacy in liquid culture are to be noted.

Based on the data shown in Table 1, bacteriophages BCP1, BCP12, BCP14and BCP37 were selected for further investigation.

Bacteriophage Propagation and Purification:

Candidate bacteriophages were propagated in liquid (broth) culture andlysates prepared from these for further work. Clarified lysates werepurified by centrifugation through a sucrose cushion (27 ml of eachlysate is carefully over-layered onto 5 ml of a sterile 10% w/v sucrose‘cushion’, in 36 ml polypropylene tubes prior to centrifugation. Thesucrose ‘cushion’ helps to remove endotoxins, while allowing the virusparticles to pellet at the bottom of the tube. Bacteriophage pelletswere resuspended in phosphate-buffered saline (PBS) and passed through a0.2 μM syringe filter to ensure sterility.

Initial Testing of Bacteriophage Mixtures:

The individual bacteriophages BCP12, BCP14 and BCP37 were then retestedboth individually at higher MOI and as a mixture, with results shown inTable 2.

The results of this testing were surprising. As can be seen from thedata shown in Table 2, bacteriophage BCP37 produced effective reductionof bacterial host numbers with very limited development of resistance.Bacteriophages BCP12 and BCP14 permitted more development of resistance.However, when a mixture of all three bacteriophages were used, whilebacterial numbers were controlled initially, the development ofresistant forms was clearly more rapid than with BCP37 alone, indicatingantagonistic effects in the mixed bacteriophage infection that permitenhanced bacterial escape.

Further testing clarified that bacteriophage BCP14 appeared to bespecifically antagonistic to the effects of bacteriophage BCP37 inreducing the development of bacterial resistance; data are shown inTable 3.

The final optical density value (OD600) given in Table 3 reflects thedevelopment of bacterial resistance after 24 hours. With mixtures of BCP37 with BCP1 or BCP12, this was greatly reduced compared to untreatedcontrols. This reduced still further when a mixture of all threebacteriophages (BCP1, BCP12, BCP37) is used. However, when bacteriophageBCP14 is used instead of BCP1, the final OD600 (and thus bacterialnumber) is markedly higher, illustrating the antagonistic effect.

Identification of Cross-Resistance:

Host bacteria that had developed resistance to the bacteriophage thatthey were treated with showed marked growth by 24 hours after infection.In order to determine whether the observed effects with initialbacteriophage mixtures were due to cross-resistance, resistant(“escape”) mutants from each assay were harvested and were treated withthe other candidate bacteriophages. This showed that resistant forms toeach of the four bacteriophages were also resistant to all of theothers; data are shown in Table 4.

Thus, all four bacteriophages (BCP1, BCP12, BCP14, BCP37) fall into thesame complementation group and allow the generation of commoncross-resistant forms of the host bacteria. It was thus desirable toidentify at least one bacteriophage which did not permit the developmentof such cross-resistance.

Evaluation of Additional Bacteriophages:

Since PAK mutants that developed resistance to individual candidatebacteriophages showed cross-resistance to other bacteriophages in thetest group, additional bacteriophages were screened to identifycandidates from existing stocks that would not be compromised by thesame resistance mechanism. Sensitivity testing identified bacteriophagesBCP6, BCP21L, BCP26, BCP28 and BCP45 as showing activity against bothBCP12-resistant and BCP37-resistant PAK mutants. The activity of thesebacteriophages against PAK in liquid culture was evaluated; data areshown in Table 5.

These results indicated that BCP28 was the most promising candidate,showing zo similar effects to BCP37 with minimal development ofresistance.

All candidate bacteriophages were then evaluated in mixtures with BCP12and BCP37; data are shown in Table 6.

Despite the limited effects of BCP6, BCP21L, BCP26 and BCP45 inindividual assays they were relatively effective in the mixtures. BCP6and BCP 28 showed the most limited development of resistance.

Given its apparent superiority in individual culture, BCP28 was selectedfor the candidate therapeutic mixture, to be combined with BCP12 andBCP37. This mixture (the three-phage mixture) thus has threebacteriophages from two complementation groups.

Final Evaluation of the Candidate Therapeutic Mixture in Vitro:

Data from the final evaluation are shown in Table 7.

Thus, a candidate mixture of three bacteriophages was identified which“flatlined” the growth of the host bacteria, producing rapid andeffective killing of the bacterial target and markedly limited thedevelopment of bacterial resistance.

In Vivo Evaluation of the Three-Phage Mix:

The three bacteriophages were purified as noted above and combined foruse in an in vivo study where infection was established using aluminescent strain of PAK (PAK-lumi).

Lytic bacteriophages with efficacy against P. aeruginosa PAK strain wereassayed in liquid cultures of host bacteria, addressing bothcross-resistance and apparent antagonism between specific bacteriophagesin the development of an optimised therapeutic mixture. Three selectedbacteriophages were mixed and used in an in vivo study where infectionwas established using a luminescent strain of PAK (PAK-lumi).

Four groups of eight BALB/C mice were infected intranasally withPAK-lumi and treated as follows:

-   All 32 mice were infected intranasally with 9×10⁶ CFU in 25 μl of    PAK Lumi in PBS-   Group 1 (n=8): Imaged and euthanized at t=2 hrs post infection-   Group 2 (n=8): Imaged and treated with PBS at t=2 hrs post infection-   Group 3 (n=8): Imaged and treated with 200 mg/kg of ciprofloxacin 2    hrs post infection in subcutaneous injection, imaged at 2, 4, 6, 8    and 24 hrs post infection and euthanized at 24 hrs post infection    (this is an extremely high dose)-   Group 4 (n=8): Imaged and treated intranasally with 30 μl of the    three-phage mix, at 2 hrs post infection, imaged at 6 and 8 hours    post infection and euthanized at 24 hrs post infection. Mice were    observed for clinical signs and infection luminescence—measured    using an IVIS in vivo imaging system. At 24 h animals were    euthanized and lung homogenate CFU/PFU determined.

Efficacy of the three-phage mix in vivo was demonstrated by bothfluorescence imaging and by enumeration of bacteria in the lung(Antibiotic=ATB=Ciprofloxacin as stated) [FIGS. 1-5].

The efficacy of the three-phage mix derived using the method aspresented was confirmed in vivo.

The bacteriophage mix showed potent activity and no resistance in vitroat 24 hours. in vivo, bacteriophage-treated mice showed a markeddecrease in luminescence after 6 h with greater reduction overallcompared with the ciprofloxacin group. This was particularly notable inthe nasopharyngeal area, although reductions were also seen inluminescence with the abdominal area. Luminescence in the lungs wasbroadly comparable, but was markedly reduced with both ciprofloxacin andthe bacteriophage mixture. By 24 h all phage and antibiotic treated micesurvived with ˜3 log. reduction in lung CFU observed for both groups.

In conclusion:

The three-phage mix is highly effective in vitro.

It is also able to rapidly control bacteria in the oropharynx and lungsof mice infected by the PAK strain of P. aeruginosa in an acute phasemodel.

Its efficacy is equivalent or superior to a high dose of an antibioticproven to be active against the infecting organism.

Its action appears to be faster than the antibiotic, and thedissemination of the infection is reduced.

Moving on from this acute model, both laboratory biofilm studies andclinical trial data from the chronically infected ear suggests that aheavily colonised, biofilm-rich environment can provide the optimalconditions for bacteriophage therapy.

The cystic fibrosis lung may provide such an environment.

Clauses

Clause 1) A method of designing a panel of bacteriophages for use as atherapeutic composition against a bacterial infection, the methodcomprising

-   -   a) assessing the activity of two or more individual        bacteriophages in separate liquid cultures, wherein each of said        separate liquid cultures consists or comprises a population of a        target bacterial species/strain (causative of the bacterial        infection) by monitoring one or more of a change in bacterial        growth rate, a development of bacterial resistance to an        individual bacteriophage, and a specificity of the development        of resistance to an individual bacteriophage;    -   b) determining the efficacy of bacteriophage combinations (e.g.        of said two or more individual bacteriophages) in a liquid        culture that consists or comprises a population of a target        bacterial species/strain (causative of the bacterial infection)        with the intention of identifying an optimised mixture of        bacteriophages, optionally by monitoring one or more of a change        in bacterial growth rate, a development of bacterial resistance        to the bacteriophage combination, and a specificity of the        development of resistance to the bacteriophage combination; and    -   c) selecting a panel of bacteriophages demonstrating        anti-microbial efficacy against the target bacterial        species/strain.

Clause 2) The method of clause 1, comprising identifying a panel ofbacteriophages having optimal anti-bacterial efficacy for therapeuticuse against the target bacterial species/strain.

Clause 3) A method for detecting cross-resistance in the method ofclause 1 or clause 2 by culturing bacteriophages in liquid cultures oftheir bacterial host by extended incubation, harvest of resistant forms,and assay against other bacteriophages specific for the same bacterialhost in liquid culture or other appropriate assay system.

Clause 4) A method for detecting antagonistic activity of bacteriophagesin clause 1 or clause 2 by culturing bacteriophages singly and inspecified combinations in liquid cultures of their bacterial host.

Clause 5) The method of clause 1 or clause 2 where activity isseparately validated in an in vivo model to confirm anti-bacterialefficacy against the target bacterial species/strain and suppression ofsaid bacterial infection.

Clause 6) The method of any preceding clause where the bacterial targetis Acinetobacter baumanii, Clostridium difficile, Escherichia coli,Klebsiella pneumonia, Pseudomonas aeruginosa, Stenotrophomonasmaltophilia, bacterial species causative of body odour, Staphylococcusaureus or Streptococcus mutans.

Clause 7) The method according to any preceding clause where thetherapeutic is for use in domestic or farm animals.

Clause 8) The method according to any preceding clause where thetherapeutic is for use in humans.

Clause 9) The method according to any preceding clause where thetherapeutic is for use in food hygiene.

Clause 10) The method according to any preceding clause where thetherapeutic is for use in agriculture or crop protection.

Clause 11) The method according to any preceding clause where thetherapeutic is for use in environmental hygiene applications.

Clause 12) A bacteriophage panel obtainable by a method according to anyof one the preceding clauses.

1. A method of designing a panel of bacteriophages as a therapeuticcomposition against a bacterial infection, the method comprising: (a)providing two or more different bacteriophages, wherein each of said twoor more different bacteriophages independently retards growth of atarget bacterial species or strain; (b) combining at least two of saidtwo or more different bacteriophages; and (c) determining growth of thetarget bacterial species or strain in the presence of said combinationof two or more different bacteriophages, wherein the target bacterialspecies or strain growth conditions are the same or equivalent in steps(a) and (c); (d) wherein, if said combination retards growth of thetarget bacterial species or strain at least equal to the greatest growthretardation achieved independently by any one of said two or moredifferent bacteriophages, the combination is accepted as a panel ofbacteriophages; and (e) wherein, if said combination retards growth ofthe target bacterial species or strain less than the greatest growthretardation achieved independently by any one of said two or moredifferent bacteriophages, the combination is initially rejected as apanel of bacteriophages.
 2. The method of claim 1, further comprising:(a) providing at least one further different bacteriophage thatindependently retards growth of the target bacterial species or strain,and (b) combining said at least one further different bacteriophage withat least one bacteriophage from a combination specified in claim 1 parts(d) and (e) to form a further combination; and (c) determining growth ofthe target bacterial species or strain in the presence of said furthercombination; (d) wherein, if said further combination retards growth ofthe target bacterial species or strain at least equal to the greatestgrowth retardation achieved independently by any one of said differentor further different bacteriophages, the combination is accepted as apanel of bacteriophages; and (e) wherein, if said combination retardsgrowth of the target bacterial species or strain less than the greatestgrowth retardation achieved independently by any one of said differentor further different bacteriophages, the combination is further rejectedas a panel of bacteriophages.
 3. The method of claim 2, wherein, in step(b), said at least one further different bacteriophage is combined withat least one bacteriophage from an initially rejected combination. 4.The method of any one of claims 1-3, wherein a panel of bacteriophagescomprises three or more different bacteriophages.
 5. A method ofdesigning a panel of bacteriophages as a therapeutic composition againsta bacterial infection, the method comprising: (a) providing a firstbacteriophage that retards growth of a target bacterial species orstrain; (b) propagating said first bacteriophage in a culture of thebacterial target species or strain until the development of bacterialresistance against said first bacteriophage, to obtain a first resistantbacterial culture; (c) providing a second bacteriophage; (d) determininggrowth of the first resistant bacterial culture in the presence of saidsecond bacteriophage; (e) wherein, if said second bacteriophage retardsgrowth of the first resistant bacterial culture, the secondbacteriophage is selected for use in a panel of bacteriophages.
 6. Themethod of claim 5, wherein, if said second bacteriophage retards growthof the first resistant bacterial culture, the combination of first andsecond bacteriophages is selected for use in the panel ofbacteriophages.
 7. The method of claim 5 or claim 6, further comprising:(a) propagating said second bacteriophage in the first resistantbacterial culture until the development of bacterial resistance againstsaid second bacteriophage to form a second resistant bacterial culture;(b) providing a third bacteriophage; (c) determining growth of thesecond resistant bacterial culture in the presence of said thirdbacteriophage; (d) wherein, if said third bacteriophage retards growthof the second resistant bacterial culture, the third bacteriophage isselected for use in the panel of bacteriophages.
 8. The method of claim7, wherein, if said third bacteriophage retards growth of the secondresistant bacterial culture, a combination of the third bacteriophageand at least one of the first and second bacteriophages is selected foruse in a panel of bacteriophages.
 9. The method of claim 7 or claim 8,wherein, if said third bacteriophage retards growth of the secondresistant bacterial culture, a combination of the first, second andthird bacteriophages is selected for use in a panel of bacteriophages.10. The method of any one of claims 1-4, wherein said two or morebacteriophages are provided by first and second bacteriophages asidentified in method claim
 5. 11. The method of any one of claims 1-4,wherein said two or more bacteriophages are provided by two or morebacteriophages selected from the first, second and third bacteriophagesas identified in method claim
 7. 12. The method of any one of claims5-10, wherein a bacterial culture is a bacterial liquid culture.
 13. Themethod of any one of claims 1-12, wherein growth is determined in abacterial liquid culture.
 14. The method of claim 13, wherein growth isdetermined in a bacterial liquid culture by measuring optical density ofthe liquid culture; preferably wherein optical density is measured at600 nm.
 15. The method of any preceding claim, wherein the bacterialtarget species or strain is selected from: Acinetobacter baumanii,Clostridium difficile, Escherichia coli, Klebsiella pneumonia,Pseudomonas aeruginosa, Stenotrophomonas maltophilia, bacterial speciescausative of body odour, Staphylococcus aureus, and Streptococcusmutans.
 16. A bacteriophage panel obtainable by a method according toany one of the preceding claims.
 17. A bacteriophage panel according toclaim 16 for use in the prevention or treatment of a bacterialinfection.
 18. The bacteriophage panel for use according to claim 17,wherein the subject is a domestic or farm animal.
 19. The bacteriophagepanel for use according to claim 17, wherein the subject is a human. 20.The bacteriophage panel for use according to any one of claims 17-19,wherein the bacterial infection is an Acinetobacter baumanii infection,a Clostridium difficile infection, an Escherichia coli infection, aKlebsiella pneumonia infection, a Pseudomonas aeruginosa infection, aStenotrophomonas maltophilia infection, a Staphylococcus aureusinfection, or a Streptococcus mutans infection.
 21. The bacteriophagepanel according to claim 16 for use in food hygiene.
 22. Thebacteriophage panel according to claim 16 for use in agriculture or cropprotection.
 23. The bacteriophage panel according to claim 16 for use inenvironmental hygiene applications.